The Ras-related GTPase superfamily contains a large number of 20-30 kDa proteins, including the Ras, Rho/Rac, Ran, ADPribosylation factor (‘ARF’) and Rab proteins. They cycle between GTP-bound and GDP-bound conformations and thereby regulate diverse cellular functions such as signal transduction, cytoskeleton organization and intracellular trafficking.
The Rab family of proteins is a member of the Ras superfamily of GTPases, or enzymes that can bind and hydrolyze Guanosine triphosphate (GTP). Approximately 70 types of Rabs have now been identified in humans. Some types of Rabs are involved in the release of neurotransmitters from synaptic vesicles.
Like other members of the Ras superfamily, Rab proteins cyclically transmit signals to downstream effectors in a guanine-nucleotide-dependent manner. In the cytosol, Rab proteins are maintained in the GDP-bound form complexed to Rab GDI (GDP dissociation inhibitor). Upon membrane association, whereas Rab GDI dissociates and is released in the cytosol, the inactive Rab protein is converted into the GTP-bound active form by GDP/GTP exchange factors (GEFs). The switch between the GDP- and GTP-bound state is essential because it determines the ability to regulate the vesicle transport machinery.
Once Rab proteins are bound to a vesicle surface, they can be activated by the replacement of guanosine diphosphate with GTP, and this exchange is catalyzed by guanine nucleotide exchange factors, or GEFs. Rabs bound to GTP are in the active conformation and can now interact with or recruit Rab effectors on target membranes within the cell. Binding of Rab to a Rab effector tethers the vesicle to its appropriate target membrane and allows other membrane surface proteins to interact, resulting in the docking of the vesicle to the target membrane. Now the Rab has fulfilled its function and the GTP is degraded to GDP (catalyzed by GTPase-activating proteins, or GAPs). The Rabs can then be recycled back to their membrane of origin. The GDP dissociation inhibitor (GDI) is necessary for the recycling pathway. This enzyme binds the prenylated Rab, inhibits the exchange of GDP for GTP (which would reactivate the Rab), solubilizes the prenyl groups, and delivers the Rab to its original membrane. GDI and REP proteins have related functions and are related enzymes. The fraction of GTP-bound Rab5 on the membrane is thus rate limiting for endosome dynamics.
Endocytosis is the process mammalian cells utilize for the uptake of essential molecules from their external environment. This material is taken into cells by membranous vesicles derived from the plasma membrane (endocytic vesicles), and once internalized, it is directed to a system of internal vesicles called endosomes. In endosomes a number of important sorting events are carried out and include the separation and segregation of the material taken into the cell from the components of the endocytic vesicle. The latter components are returned (recycled) to the plasma membrane for use in subsequent rounds of endocytosis, whereas the endocytosed material is mostly sorted into lysosomes. The endosomal system is composed of a series of distinct elements, some of which are thought to perform sorting (rab5-positive endosomes) or recycling (rab11-positive endosomes) activities. Late endosomes (rab7-positive endosomes) appear to participate in routing material destined for degradation from the rab5-positive sorting compartment to the lysosomal compartment.
Rab5 is a small GTPase localized on early endosomes, and Rab5 controls early endosome fusion along the endocytic pathway. Rab5 functions as a molecular switch regulating endocytosis by facilitating early endosome fusion and possibly the budding of endocytic vesicles from the plasma membrane as well. Overexpression of Rab5 in cultured cells stimulates endocytosis. For newly synthesized Rab5 molecules to obtain biological activity, they first need to be modified by isoprenylation, then escorted to endosomal membranes where they undergo nucleotide exchange to become the active GTP-bound conformation. These processes have been suggested to be facilitated by Rab5-specific membrane recruitment machinery and a guanine nucleotide exchange factor. The activated Rab5 recruits effectors to promote the docking of endosomes, which ultimately leads to membrane fusion.
A number of different cytosolic factors have been identified in mammalian cells that are effector molecules that interact with Rab proteins with specificity for the triphosphate conformation. While it is intriguing that all these proteins share no detectable sequence homology, they all appear to be soluble factors that can be recruited on the membrane by the active form of Rab proteins. Examples of such molecules which act as effectors for Rab5 include, but are not limited to, Rabaptin-5, EEA1 (early endosome autoantigen), APPL1 and APPL2 (adaptor protein containing PH domain, PTB domain, and Leucine zipper motif 1 or 2), and Rabenosyn-5.
Prior to the present invention, the only way to determine Rab5 activity in a cell was to label the cell with [32P]orthophosphate, followed by immunoprecipitation with Rab5 antibodies, thin-layer chromatography, and autoradiography. This is a difficult procedure that requires a lengthy amount of time (at least 6 hours) to complete, and also utilizes hazardous radioactive reagents.
Therefore, new and improved methods of determining intracellular Rab5 activity are being sought that are faster, easier to perform and safer than the prior art methods. It is to such assays that the present invention is directed.